The long-range goal of the proposed research is to elucidate the mechanism (s) by which 17 -estradiol (E2) signals in hypothalamic neurons to control homeostasis. It has been suggested that the classical estrogen receptors ER and ER, and possibly different splice variants, acting through rapid and long term genomic mechanisms are responsible for all of the actions of E2 in the central nervous system (CNS). However, our studies indicate that a novel membrane E2 receptor (mER) is involved in a rapid signaling cascade that leads to increased excitability and new gene transcription in hypothalamic proopiomelanocortin (POMC) neurons. We have developed a mER selective ligand STX, and both E2 and STX are fully efficacious in estrogen receptor , and GPR30 knockout mice. Moreover as proof of principle, in vivo treatment with STX, similar to E2, attenuates the weight gain in hypo-estrogenic female guinea pigs. Although it is known that leptin and insulin inputs to POMC and neuropeptide Y (NPY) neurons are important for energy homeostasis, the cellular mechanism (s) by which E2 modulates leptin and insulin signaling in anorexigenic POMC and orexigenic NPY neurons is relatively unexplored. Further studies are needed to characterize the cross-talk of the mER with other hormone signaling pathways in POMC neurons and its role in modulating the excitability of other hypothalamic (NPY) neurons involved in the control of energy homeostasis. Therefore, our current work focuses on hypothalamic arcuate POMC and NPY neurons in which E2 acts through multiple signaling cascades. Our multidisciplinary approach incorporates a unique range of cellular and molecular tools and our combined expertise (i.e., electrophysiology, chemistry, molecular biology, histochemistry and whole animal physiology). Our working hypothesis is that the anorexic effects of E2 are due, in part, to the activation of a mER in addition to ER (and ER) in POMC and NPY neurons that ultimately up regulates POMC and down-regulates NPY excitability and gene expression. In addition, we hypothesize that estrogen signaling pathways act in concert with leptin and insulin signaling pathways in these anorexigenic and orexigenic neurons. Therefore, our specific aims are the following: (1) to elucidate the effects of E2 and STX on leptin signaling in POMC neurons; (2) to characterize the effects of E2 and STX on insulin and leptin signaling in NPY neurons; (3) to elucidate the POMC input to NPY neurons; and (4) to characterize leptin and insulin signaling in guinea pig POMC and NPY neurons during the early and late follicular phases of the ovulatory cycle. Understanding these novel actions of E2 will provide critical insight into the fundamental role of E2 withdrawal in menopausal states and allow the further development of new estrogenic compounds specifically targeting these critical brain circuits involved in the control of homeostasis. PUBLIC HEALTH RELEVANCE: In spite of the controversy with hormone replacement therapy, it is known that 17-estradiol (E2) maintains multiple autonomic functions including energy and temperature homeostasis, sleep-wake cycles (circadian rhythms) and positive mood and affect. Consequently, the availability of selective estrogen receptor modulators that elicit the beneficial effects of estrogen in the CNS but lack its peripheral risks and unwanted side effects is of considerable clinical interests. We have developed a selective estrogen receptor modulator that targets a membrane E2 receptor (mER) in hypothalamic neurons. The current studies will characterize the role of the mER signaling pathway in modulating the leptin and insulin signaling pathways in hypothalamic neurons involved in controlling energy homeostasis. Understanding these novel actions of E2 and how they relate to its genomic actions will provide insight into the fundamental role of E2 withdrawal in menopausal states and also in pathological states such as polycystic ovarian syndrome in which there is a high incidence of insulin resistance, diabetes and obesity.